One of the important protein functions includes a function specifically binding to a specific molecule. This function plays an important role in immunoreaction and signal transduction in the body. Technologies using the function to separate and purify useful substances are actively developed. One example actually used for industrial purposes includes a protein A affinity separation matrix (hereinafter, protein A may be abbreviated as “SpA”) which is used to purify (capture) an antibody medicine from an animal cell culture with a high purity at a time (Non-Patent Documents 1 and 2).
Antibody medicines which have been developed so far are generally monoclonal antibodies. These antibodies are mass-produced by using recombinant cell culture technology or the like. The “monoclonal antibodies” refer to antibodies produced by clones of a single antibody-producing cell. Almost all antibody medicines currently available on the market are classified into immunoglobulin G (IgG) subclasses based on their molecular structures. Further, clinical development of antibody medicines composed of antibody derivatives having molecular structures of fragmented immunoglobulins (fragment antibodies) has been actively conducted. A plurality of antibody medicines composed of immunoglobulin Fab fragments has been available on the market (Non-Patent Document 3).
In an initial purification step in antibody medicine production process, the above-described SpA affinity separation matrix is used. However, SpA is generally a protein that specifically binds to an Fc region of IgG. Accordingly, fragment antibodies that do not include the Fc region cannot be subjected to capturing using the SpA affinity separation matrix. Therefore, from the viewpoint of making the antibody medicine purification process into a platform, there are great industrial needs for the affinity separation matrix capable of capturing fragment antibodies that do not include the Fc region of IgG.
A plurality of proteins that bind to regions other than the Fc region of IgG has been already known (Non-Patent Document 4). However, there is no fact that the affinity separation matrix using such proteins as ligands is used for standard industrial purposes of purification of the antibody medicine, similarly to the SpA affinity separation matrix.
For example, a protein called protein G found in group G of Streptococcus sp. (hereinafter, protein G may be abbreviated as “SpG”) has a property of binding to IgG. Also, there is an SpG affinity separation matrix product having SpG immobilized thereon as a ligand (product name: “Protein-G Sepharose 4 Fast Flow”, manufactured by GE Healthcare, Patent Document 1). It is known that SpG strongly binds to the Fc region of IgG, but weakly binds to the Fab region (Non-Patent Documents 4 and 5). However, since the binding force of SpG to the Fab region is weak, it can be said that the SpG affinity separation matrix product has a low retention performance for fragment antibodies that do not include the Fc region, but include only the Fab region. Consequently, an attempt is being carried out to improve the binding force to the Fab region by introducing a mutation into the SpG (Patent Document 2).